Cytochrome P450 is a heme-containing protein which was discovered by its unusually reduced carbon monoxide difference spectrum that has an absorbance at 450 nm, which is caused by a thiolate anion acting as the fifth ligand to the heme. The most common reaction catalyzed by cytochrome P450 is hydroxylation, often of a lipophilic substrate. Thus, cytochrome P450 proteins are frequently called hydroxylases. However, cytochrome P450 proteins can perform a wide spectrum of reactions including N-oxidation, sulfoxidation, epoxidation, N-, S-, and O-dealkylation, peroxidation, deamination, desulfuration, and dehalogenation.
In bacteria, the P450 proteins are soluble and approximately 400 amino acids long. In eukaryotes, P450 proteins are larger, being about 500 amino acids. In eukaryotes, the proteins are usually membrane bound through an N-terminal hydrophobic peptide and other less well understood contacts. The two locations of P450 in eukaryotes are the endoplasmic reticulum membrane and the mitochondrial inner membrane, which, collectively, are referred to as “microsomes.”
There are more than 1500 known P450 sequences which are grouped into families and subfamily. The cytochrome P450 gene superfamily is composed of at least 207 genes that have been named based on the evolutionary relationships of the cytochromes P450. For this nomenclature system, the sequences of all of the cytochrome P450 genes are compared, and those cytochromes P450 that share at least 40% identity are defined as a family (designated by CYP followed by a Roman or Arabic numeral, e.g., CYP3), and further divided into subfamilies (designated by a capital letter, e.g., CYP3A), which are comprised of those forms that are at least 55% related by their deduced amino acid sequences. Finally, the gene for each individual form of cytochrome P450 is assigned an Arabic number (e.g., CYP3A4).
CYP3A isoenzyme is a member of the cytochrome P450 superfamily which constitutes up to 60% of the total human liver microsomal cytochrome P450 and has been found in alimentary passage of stomach and intestines and livers. CYP3A has also been found in kidney epithelial cells, jejunal mucosa, and the lungs. CYP3A is one of the most abundant subfamilies in cytochrome P450 superfamily.
At least five (5) forms of CYPs are found in human CYP3A subfamily, and these forms are responsible for the metabolism of a large number of structurally diverse drugs. In non-induced individuals, CYP3A may constitute 15% of the P450 enzymes in the liver; in enterocytes, members of the CYP3A subfamily constitute greater than 70% of the CYP-containing enzymes.
The first two (2) CYP3A subfamily members identified were CYP3A3 and CYP3A4. These two (2) CYP3As are so closely related that the majority of studies performed to date have not been able to distinguish their contributions, and thus, they are often referred to as CYP3A3/4. The levels of CYP3A3/4 vary in human liver samples.
CYP3A is responsible for metabolism of a large number of drugs including nifedipine, macrofide antibiotics including erythromycin and troleandomycin, cyclosporin, FK506, teffenadine, tamoxifen, lidocaine, midazolam, triazolam, dapsone, diltiazem, lovastatin, quinidine, ethylestradiol, testosterone, and alfentanil. CYP3A3/4 involves in erythromycin N-demethylation, cyclosporine oxidation, nifedipine oxidation, midazolam hydroxylation, testosterone 6.-β.-hydroxylation, and cortisol 6.-β.-hydroxylation. CYP3A has also been shown to be involved in both bioactivation and detoxication pathways for several carcinogens in vitro.
In recent years, CYP3A has been proven to be the responsible for the first-pass effect of drug degradation. Because CYP3A inhibitors inhibit the CYP3A enzymatic activity, they have the capacity of improving the bioavailability of the drugs. In addition, an effective CYP3A inhibitor can bind to CYP3A and thus decrease the clinical interaction between drugs induced by CYP3A. Furthermore, CYP3A has been shown to be responsible for transforming Alfa toxin B1 and Alfa toxin G1 into carcinogen. Thus, CYP3A inhibitors can also serve as chemopreventors.
Because an effective CYP3A inhibitor has the capability of improving the bioavailability for drugs, decreasing certain clinical drug interactions induced by CYP3A, and acting as a chemopreventor, an effective CYP3A inhibitor is well sought after in the field of medical science.
So far, there have been reports relating to the improvement of drug bioavailability. For example, U.S. Pat. Nos. 5,716,928, 5,665,386, 5,916,566, and 6,121,234 and, describe essential oil or essential oil components as regulators to improve drug bioavailability. However, no correlation or connection between the improvement of drug bioavailability and the inhibition of CYP, particularly CYP3A, is provided. Also, U.S. Pat. No. 6,063,809 correlates citrus-derived substances (such as grapefruit juice) to drug bioavailability due to anti-first-pass effect regulated by cytochrome P450.
There are also some publications which connect the improvement of drug bioavailability to inhibition of CYP3A activity. For examples, U.S. Pat. No. 5,962,522 describes the improvement of drug bioavailability by propyl gallate, which inhibits CYP3A enzymatic activities; U.S. Pat. No. 6,004,927 discloses certain naphthalenes and flavonoids, which reduce CYP3A drug biotransformation in the gut by acting either as an inhibitor of CYP3A activity or as a substrate of CYP3A activity; U.S. Pat. No. 6,028,054 discloses certain compounds, including flavonoids, inhibit CYP3A; and, Sarkar, Cancer Chemother. Pharmacol., (1995), 36: 448–450, discloses that quercetin inhibits CYP3A isozymes.
In addition, there are reports relating to the regulation of CYP3A. For example, Hukkanen et al., Am. J. Respir. Cell. Mol. Biol. (2000), 22: 360–6, disclose the induction of CYP3A5 by dexamethasone and phenobarbital in human alveolar type II cell-derived A549 adenocarcinoma cell line and by glucocorticoid in human lung cells.
Finally, there have been reports that certain chemical compounds can induce CYP3A expression or activity. For example, Backlund et al., J. Biol. Chem. (1997), 272: 31755–63, disclose that omeprazole increases CYP3A expression in rat hepatoma H4IIE cell line; Paolini et al., Cancer Lett. (1999), 145: 35–42, disclose that glycyrrhizin induces CYP3A in Sprague-Dawley rat liver monooxygenase; Paolini et al., Life Sci. (1998), 62: 571–82, disclose the induction of hepatic CYP3A in murine liver by glycyrrizin; Ronis et al., Biochem. Pharmacol. (1994), 48: 1953–65, disclose that clotrimazole induces CYP3A isozyme expression in male Sprague-Dawley rat and the male bobwhite quail; Backman et al., Clin. Pharmacol. Ther. (2000), 67: 382–90, disclose that tangeretin, a flavonoid, stimulates the catalytic activity of CYP3A4 in human liver microsomes.
In the invention to be presented in the following sections, various CYP3A inhibitors and enhancers are tested. These inhibitors and enhancers are natural compounds extracted from herbs which shows no sign of toxic effects. The CYP3A inhibitors inhibit the CYP3A enzymatic activity so as to improve the bioavailability of certain drugs in vivo. The CYP3A enhancers induce the CYP3A enzymatic activity so as to improve drug biotransformation by CYP3A, resulting in removal of active drugs. Thus, CYP3A inhibitors can be used as anti-first-pass effect compounds to improve the bioavailability of certain drugs or as chemopreventors by preventing the conversion of compounds into carcinogen caused by CYP3A. In addition, CYP3A enhancers can be used to improve biotransformation and elimination of active drugs or CYP3A substrates.